1. Field of the Invention
This invention relates to a cytotoxic agent directed against tumor cells. In particular, the invention is directed to a cytotoxin derived from a macrophage cell line.
2. Description of Related Art
Activated macrophages become cytotoxic to tumor cells in response to various biological and microbial signals. Inhibition of mitochondrial respiration, loss of intracellular iron, and loss of aconitase activity by L-arginine-dependent pathways, have been shown to result from macrophage cytotoxic activity against tumor cells.
Recent studies suggest that one mechanism of macrophage-mediated cytotoxicity is L-arginine dependent via the oxidative degradation of L-arginine to nitric oxide (NO). Thus, NO has been proposed to be a chemically reactive component that leads to tumor cell mitochondrial respiration inhibition, loss of intracellular iron, inhibition of aconitase activity, and inhibition of DNA synthesis. NO is probably an active intermediate as established by the following criteria: (a) macrophages, upon activation, release nitrite and nitrate (NO) in an L-arginine-dependent manner; (b) the production of NO by macrophages has been directly demonstrated; and (c) NO alone has been shown to mimic some cytotoxic effects attributed to activated macrophages. [Hibbs et al., L-Arginine is Required for the Expression of the Activated Macrophage Effector Mechanism Causing Selective Metabolic Inhibition of Target Cells, J. Immunol. 138:550-565 (1987) and Stuehr et al., Nitric Oxide: A Macrophage Product Responsible for Cytostasis and Respiration Inhibition in Tumor Target Cells, J. Exp. Med., 169:1543-1545 (1989)].
Studies from our laboratory as well as others (Amber et al., J. Leukoc. Biol. 43; 187-192 (1988) and Amber et al., J. Leukoc. Biol. 44:58-65 (I988)) have shown that the L-arginine-dependent nitric oxide effector mechanism, is probably mediated by a combination of interferon-gamma with TNF, IL1 or LPS present in the conditioned supernatants. This conclusion was derived from the following observations. 1) significant amounts of nitric oxide were produced in EMT-6 cells upon treatment with macrophage conditioned supernatants and 2) the mitochondrial respiration inhibition was directly proportional to the amount of L-arginine (from which nitric oxide is derived) present in the culture medium and 3) the respiration inhibition and nitrite production were probably caused by a combination if IFN-.gamma. and TNF present in the conditioned supernatants since the bioactivity was almost completely neutralized by anti-interferon-.gamma. (78%) and anti-TNF antisera (11%), shown by in vitro experiments (Amber, et al, J. Leukoc. Biol. 46:307 (1989)).
One study has stated that L-arginine deficient environments enhance resident macrophage metabolism, functions, and viability (Albina et al., An L-Arginine (ARG) Deficient Environment Enhances Resident Macrophage Metabolism, Functions and Viability, Fed. Am. Soc. Exp. Biol. J., 3(4), A966 (March 1989)). This study involved measuring increased macrophage viability and therefore did not focus on the cytotoxicity contained in the supernatant collected from the cultured macrophages.
Another reference disclosed experiments which employed conditioned supernatant collected from activated murine peritoneal macrophages. The authors demonstrated that conditioned supernatant, obtained from activated macrophages, contained a component that inhibited tumor cell mitochondrial respiration. Mitochondrial respiration was measured by EMT-6 cell's ability to oxidize succinate, malate, and tetramethylphenylenediamine (Kilbourn et al., Activated Macrophages Secrete A Soluble Factor That Inhibits Mitochondrial Respiration of Tumor Cells, J. Immunol., 133:2577-2581 (1984); Kilbourn, R. G., Inhibition of the Mitochondrial Respiration of Tumor Cells by Soluble Factors Released by Activated Macrophages, A Ph.D. Dissertation presented to the Faculty of The University of Texas Health Science Center at Houston Graduate School of Biomedical Sciences [May, 1984], Dissertation Abstracts International, 45(5):1424B-1425B, November, 1984). Kilbourn et al (1984) also reported that the conditioned supernatant inhibited DNA synthesis in EMT-6 cells (used to measure the level of cytostasis induced in the target cells). Fractionation of conditioned supernatant by molecular exclusion resulted in the recovery of two peaks that both demonstrated respiration inhibition activity. The authors demonstrated that these two peaks, eluting at 55,000 and 80,000 daltons, mediated the inhibition of malate and succinate oxidation and were cytostatic for EMT-6 cells. The peritoneal macrophage conditioned supernatant described by Kilbourn et al. (1984) are obtained under conditions very similar to that described by Amber et al. (Amber et al., J. Leukoc. Biol. 43:187-192 (1988)) and therefore the observed mitochondrial respiration inhibition is very likely to be mediated via the nitric oxide pathway by IFN-.gamma. and potentially by a combination of other cytokines.
The present invention provides a cytokine that acts via a different mechanism than earlier described macrophage cytotoxin.
This present invention therefore discloses a method for producing a cytokine that causes cytostasis and mitochondrial respiration inhibition independent of nitrite production. This cytokine is obtained by culturing an independently established macrophage cell line (EA13.5), adding IFN-.gamma. and lipopolysaccharide to trigger the secretion of the macrophage cytotoxin into the media, allowing its isolation.